Ligand activation of the receptors in the plasma membrane initiates cell signaling which propagates into various intracellular compartments through multi-step processes that depend on cellular second messengers. It has been shown that these short lived small molecules not only amplify the signal in the relay but also confer specificity by their temporal and spatial distribution in the cell.
Although a relatively minor component of cellular membranes, phosphoinositides (PIs) are emerging to be a crucial component and a diverse family of lipid second messengers (1). The family's diversity stems from the existence of multi-phosphorylated states at the D-3, -4 and -5 positions, yielding seven distinct signaling molecules identified to date (2). In particular, phosphatidylinositol 3-kinase (PI3K) synthesizes four species of D-3 phosphorylated inositides, namely phosphatidylinositol 3,4,5 triphosphate (PIP3; also known as “PI(3,4,5)P3”), PI(3,4)P2, PI(3,5)P2 and PI(3)P (3). On the other hand, they are dephosphorylated by the 3′-phosphatase called phosphatase and tensin homologue deleted on chromosome ten (PTEN) and some by 5′-phosphatases (4, 5). PIP3 and PI(3,4)P2 are responsible for the recruitment of the serine/threonine kinase Akt, also known as and referred to herein as protein kinase B (PKB), to the plasma membrane where phosphorylation at two sites, T308 and S473 (in Akt1), fully activates the kinase (6, 7).
Upon activation Akt plays important roles in various cellular processes such as proliferation, differentiation, survival, and tumorigenesis (8). For example, activation of Akt by insulin or growth factors is prevented if the cells are preincubated with inhibitors of PI 3-kinase, the best known being Wortmannin or LY 294002, or by overexpression of a dominant negative mutant of PI 3-kinase (44). Further, mutation of the tyrosine residues in the PDGF receptor that when phosphorylated bind to PI 3-kinase also prevent the activation of PKBα, an isoform of PKB. Recent reports have shown the PKB is itself activated by another kinase also downstream of PIP3 (45). This kinase, termed PKB kinase, or phosphatidylinositide (PtdIns) 3-kinase (PDK1), requires PIP3 for activation (46).
Akt also is involved in regulating cell growth. It has been implicated in certain human cancers; for instance, it is known to be amplified in a percentage of ovarian carcinomas, breast carcinomas, and pancreatic carcinomas (47; 48). The amplification of the enzyme affords tumor cells a mechanism to circumvent apoptosis. Drugs that inhibit PKB activity are useful for treating diseases involving inappropriate cell growth, including cancer.
Due to the involvement of these PIs in such diverse functions, a cell must precisely control their spatial and temporal dynamics to avoid abnormalities, yet there are no reliable methods available for measuring PIP3, PI(3,4)P2 dynamics within subcellular compartments with high spatiotemporal resolution. Previously employed indicators cause artifacts due to cell movements and additionally cannot be targeted to subcellular locations. Biochemical methods of radiolabeling and cell fractionation measure total PI levels and have poor spatial and temporal resolution, and use of specific antibodies (31) to immunodetect PIs in fixed cells provides only snapshots at different time points. For dynamic monitoring of the lipid molecules, live-cell imaging using PH domains fused with fluorescent proteins (10-13, 32, 33) has been widely used, however their dependency on translocation limits their abilities to examine different pools of PIs.
There are ratiometric sensors for monitoring PI dynamics. On such sensor, termed “fllip” (34), employs a PH domain of GRP1 and required membrane anchoring to facilitate a PIP3 binding induced conformational change via rotation of rigid linkers around a diglycine hinge engineered within the construct, which limits the targetability of the reporter and generalizability of the design. Another ratiometric sensor, termed “CAY,” is based on a peptide from Listeria protein ActA that undergoes a random coil to helix transition upon lipid binding (35). CAY is a sensor for polyphosphorylated PIs showing some preference for PIP3 in cells.
There is a need in the art for sensitive, targetable indicators that would permit direct measurement and assessment of specific signaling of 3′-phosphoinositides.